Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.     

N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin

A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N-monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.     

Balance of apoptotic cell death and survival in allergic diseases

Allergic diseases result from over-reaction of the immune system in response to exogenous allergens, where inflammatory cells have constantly extended longevity and contribute to an on-going immune response in allergic tissues. Here, we review disequilibrium in the death and survival of epithelial cells and inflammatory cells in the pathological processes of asthma, atopic dermatitis, and other allergic diseases.     

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5 years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P < 0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride + total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin

In this study, the effect of puerarin on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.5mM H(2)O(2)may cause significant viability loss and apoptotic rate increase. When c-Myc, Bcl-2 and Bax expression and caspase-3 activity were measured, using Ac-DEVD-AMC as a substrate, the changes in these apoptosis regulatory and effector proteins suggested that the elevation of c-Myc, decrease in Bcl-2:Bax protein ratio, and caspase-3 activation all play a key role in apoptosis. When cells were treated with puerarin prior to 0.5 mM H(2)O(2)treatment, a reduction in viability loss and apoptotic rate was seen. In addition, c-Myc expression decreased and Bcl-2:Bax ratio increased. Puerarin also reduced the H(2)O(2)-induced elevation of caspase-3 activation. These results suggest that puerarin can protect neurons against oxidative stress. It can block apoptosis in its early stages via the regulation of anti- and pro-apoptotic proteins, as well as by the attenuation of caspase-3 activation in H(2)O(2)-induced PC12 cells.     

Distinct nuclear factor-kappaB/Rel proteins have opposing modulatory effects in glutamate-induced cell death in HT22 cells

Members of the nuclear factor-κB (NF-κB)/Rel family (p50, p52, p65 (RelA), RelB and c-Rel) is sequestered in the cytoplasm through its tight association with the inhibitor of NF-κB (IκB). NF-κB has been shown to function as key regulators of either cell death or survival in neurons after activation of the cells by various extracellular signals. In the study presented here, we investigated whether the selective activation of diverse NF-κB/Rel family members in HT22 cells might lead to distinct effects on glutamate-induced cell death. Exposing HT22 cells to glutamate, which blocks cystine uptake into the cells via inhibition of the glutamate-cystine antiporter, resulted in a transient activation of IκB and NF-κB/Rel and caused delayed cell death. Aspirin, which has been shown to block phosphorylation of the IκB component of the cytoplasmic NF-κB complex, significantly suppressed glutamate-induced cell death, whereas the NF-κB decoy oligonucleotide potentiated it. The inhibition of NF-κB/Rel protein expression by antisense oligonucleotides showed that p65 is involved in glutamate-mediated cell death, whereas p50 is involved in inhibitory pathways of the cell death. These findings suggest that in HT22 cells, the balance between promoting and presenting cell death to glutamate-induced oxidative stress relies on the activation of distinct NF-κB proteins.     

Effects of boric acid on cell death and oxidative stress of mouse TM3 Leydig cells in vitro

BackgroundBoron (B) is an abundant element on earth and presents at physiological pH in the form of boric acid (BA). It has both positive and negative effects on biological systems. BA and sodium borates have been considered as being toxic to the reproduction system in animal experiments. Unfortunately, the molecular mechanism underlying the toxic effects of BA is not fully understood.MethodsHere, we demonstrate the influence of BA on mouse TM3 Leydig cells which are male reproductive system cells targeted by BA exposure. The cytotoxicity was evaluated by MTT and NRU assays. Annexin V-FITC/PI double staining kit, mitochondria membrane potential (ΔΨm) assay kit with JC-1 and caspase-3 colorimetric assay kit were used to indicate the cell death pathway. To estimate the role of oxidative stress in BA induced toxicity, glutathione (GSH) level, catalase (CAT) and superoxide dismutase (SOD) activities were measured manually.ResultsThe cell viability assays showed that BA was not cytotoxic within the tested concentrations up to 1000 μM. Sub-toxic concentrations were used for detecting oxidative stress status. BA exposure was significantly reduced GSH level at 1000 μM and CAT activity in a concentration-dependent manner. However, SOD activity was increased at the tested concentrations (100–1000 μM). Moreover, ΔΨm was significantly decreased at 500 and 1000 μM of BA, while caspase-3 activity was not changed apparently.ConclusionThese findings demonstrated that BA is not cytotoxic and apoptotic but may slightly induces oxidative stress in TM3 Leydig cells at higher concentrations.     

Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Protective effect of vanillic acid in hydrogen peroxide-induced oxidative stress in D.Mel-2 cell line

The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H₂O₂). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H₂O₂-induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H₂O₂ and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC₅₀ value of vanillic acid and H₂O₂ was found 250 μg/ml and 125 μg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H₂O₂ treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H₂O₂ treated group. The effect of H₂O₂ on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H₂O₂ treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.